The present invention relates to a plant into which DNA encoding a basidiomycete-derived peroxidase is transferred, and to a method for decomposing and removing hazardous chemicals using said plant.
Physical and chemical treatments have been developed as technologies to treat the industrially produced chemical substances, PCB, BHC and DDT, or the unintentional product, dioxin, when they are present or accumulated at high concentration in an environment. For example, techniques such as photochemical decomposition, supercritical water decomposition, solvent extraction decomposition, catalytic oxidation, vapor phase hydrogenation reduction, melt combustion, heat treatment in reducing atmosphere, and glassification treatment, have been experimentally tested. However, those physical and chemical treatments seem to be impractical in view of cost-efficiency for hazardous chemicals accumulated at low levels in the environments as in soils or rivers, and moreover, in-situ treatments are required. Even when the concentration of these extensively diffused substances is low, the level is enough for endocrine disturbance. As a means to overcome this problem, bio-remediation has been performed with the use of microorganisms that strongly decompose hazardous chemicals. Such decontamination by microorganisms also has problems. That is, to maintain such microorganisms predominantly over the long term, the inoculation of microorganisms and application of nutrients are essential, which becomes more difficult as the area of contamination expands.
Under these circumstances, an attempt has been made recently for decontamination by phyto-remediation (restoration of the environment by plants) using plants.
Plants can be grown independently taking nourishment from the sun, water, and inorganic ions, and can be cultivated extensively by controlling seeds. Because of this, plants have attracted attention as a method for long-lasting environmental decontamination.
Studies on phyto-remediation to agricultural chemicals (Plant physiol, 106, 17, 1994), heavy metals (Trends Biotech, 13, 393, 1995), organic mercury (Nature Biotechnol, 16, 925-928, 1998), selenium (Plant Physiol, 119, 565-573, 1999), MTBE of a gasoline additive (Soil and Groundwater Cleanup February/March, 42-45, 1999), nitrogen oxides (Plant, Cell and Environment, 21, 180-190, 1998), and radioactive compounds (Science, 277, 515-522, 1997), have been made so far. Further study concerns the remediation of contaminated soils by cooperation of plants and rooting zone microorganisms comprising enhancing growth of soil microorganisms or an improvement of flora by cultivating plants in contaminated soils.
The phyto-remediation that has been examined, includes use of the detoxification mechanism or evaporation ability which plants originally bear. Moreover, an attempt has been made to enhance the environmental decontamination function of plants by transferring genes from microorganisms.
The environmental remediation by gene recombinant plants, in the case of for example organic mercury, is carried out by reducing the organic mercury to a metal mercury and vaporizing it in the atmosphere. In the case of agricultural chemicals or heavy metals, the remediation is carried out by transport to and accumulation in cell wall fraction. However, because the accumulated environmental contaminants are released again in the environment through blighting of plants, said remediation does not contribute to fundamental decontamination. Furthermore, in the case of dioxin or PCB, it is predictable that readily degradable substances are decomposed while non-degradable and highly toxic substances are condensed and accumulated. Therefore, it is thought that conventional phyto-remediation is insufficient.
As detailed above, an attempt to decompose hazardous chemicals directly in plant cells using recombinant plants into which an enzyme gene for decomposing hazardous chemicals derived from microorganisms is transferred, has been made with respect to the decomposition of 2,4,6-trichlorophenol (Japan Society for Bioscience, Biotechnology, and Agrochemistry, Abstracts for the Annual Meeting, p146, 1998) or xcex3-hexachlorocyclohexane (Japan Society for Bioscience, Biotechnology, and Agrochemistry, Abstracts for the Annual Meeting, p89, 1997).
Incidentally, the basidiomycete-derived peroxidase gene has two types of families, lignin peroxidase gene (LiP) and manganese peroxidase gene (MnP). These genes were isolated from various basidiomycetes, such as Phanerochaete chrysosporium, Phlebia radiata, Coriolus and Pleurotus, and the primary structures thereof were determined (Nature, 1987. 326, 520-523, Nucelic Acid Res, 1988, 16, 1219, Gene, 1989, 85, 343-351, Biochem Biopys Res Commun, 1991, 179, 428-435, J Biol Chem, 1989, 264, 5036-5040, Biochim Biophys Acta, 1995, 1263, 71-74, Biochim Biophys Acta, 1251, 205-209). It has been demonstrated that enzymes encoded by these genes decompose a variety of chemical substances which are not readily degradable. The enzymes oxidatively decompose endocrine-disturbance substances including polycyclic aromatic hydrocarbons (Biodegradation, 1999, 10, 159-168, Appl Environ Microbiol, 1996, 1597-1603), chlorophenols (Biochem Biophys Res Commun, 1999, 257, 901-905, J Bacteriol, 1998, 180, 5159-5164) and dioxin (BioEssays, 1986, 6, 166-170, J Bacteriol, 1992, 174, 2131-2137), etc.
As described above, the basidiomycete-derived peroxidase oxidatively decomposes various hazardous chemicals; therefore, if a plant capable of exerting the effect of this enzyme could be prepared, it would be very useful for decomposing and removing hazardous chemicals in the environment.
However, the preparation of such plants is predicted to be difficult for the following reasons.
1) The production of a basidiomycete-derived peroxidase as a stable and active protein in plant cells is known to be difficult because of high GC content of the peroxidase gene and the codon usage, and there are no successful examples.
2) Even if the difficulty described in 1) is overcome, it was predicted to be difficult to produce a plant capable of decomposing and removing hazardous chemicals in the environment effectively, because in general, environmental contaminants are highly fat-soluble (or lipophilic) chemical compounds, and because for example PCB or dioxin is absorbed from the roots of plants and the amount taken-up within plant cells is very small. Furthermore, it was estimated that obtaining transformed plants capable of effectively decomposing and removing chemical compounds as described above was difficult unless the function of the peroxidase is expressed on the cell surface of plant roots.
3) Generally, hydrogen peroxide is considered to exist on the surface layer of plant cells. However, even when transformed plants with functions described in 2) can be obtained, the reaction system for decomposition of hazardous chemicals is difficult to make it to function unless the production and distribution of hydrogen peroxide in the plant and the basidiomycete-derived peroxidase produced by gene transfer, are synchronized spatially and temporally.
Under the technical backgrounds described above, an object of the present invention is to provide a plant capable of decomposing and removing hazardous chemicals in the environment effectively.
The inventors have gained the following findings after extensible and intensive studies for solving above problems.
1) Since the basidiomycete-derived peroxidase produced by gene transfer exists in an active and stable form on the cell surface of transgenic plant roots, hazardous chemicals are decomposed by the peroxidase in the rooting zone even when the peroxidase is not incorporated within cells.
2) Since endogenous hydrogen peroxide exists in plant bodies and is released from roots synchronously with production of the peroxidase, hazardous chemicals can be decomposed without adding hydrogen peroxide externally.
The present invention has been completed based on the above findings.
More specifically, the present invention relates to a plant into which DNA encoding the basidiomycete-derived peroxidase is transferred, said DNA being expressed therein.
Furthermore, the present invention relates to a method for decomposing and removing hazardous chemicals, comprising cultivating the above plant in the environment contaminated with hazardous chemicals.